Treatment of demyelinating diseases

ABSTRACT

Described herein are methods of promoting remyelination in a subject suffering from demyelination diseases by administering to the subject a combination of steroid hormones and Hedgehog signaling pathway modulators. Also described are methods of administering the combination of drugs, wherein the combination of drugs are in compositions adapted for nasal administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/IB2019/050198, filedJan. 10, 2019, and claims priority under 35 U.S.C. § 119 to U.S.provisional application 62/616,173 filed Jan. 11, 2018, the entirecontents of which are incorporated herein by reference.

FIELD

Described herein are compositions and methods for treating demyclinatingdiseases using a steroid hormone and a Sonic hedgehog signaling pathwaymodulating agent.

BACKGROUND

Myelin is a fatty white substance that surrounds the axon of some nervecells, forming an electrically insulating layer. In humans, around 40%of the brain contains white matter comprising densely packed fibers, ofwhich myelin is a main component (50-60% dry weight of the whitematter). Myelin is synthesized and maintained by oligodendrocytes (OLs)in the central nervous system (CNS). Oligodendrocytes are a type ofneuroglia that function to provide support and insulation to axons inthe CNS. Oligodendrocytes are generated from oligodendrocyte precursorcells (OPCs), and are found only in the CNS.

In demyclinating diseases, the myelin sheath of neurons of the nervoussystem is damaged. This damage may impair the conduction of signals inthe affected nerves, leading to, for example, deficiency in sensation,movement, cognition, and other functions, depending on the nervesinvolved. Among the numerous demyelination diseases, multiple sclerosis(MS) is the most widespread disabling neurological condition of youngadults around the world. The Multiple Sclerosis Foundation estimatesthat more than 400,000 people in the United States and about 2.5 millionpeople around the world have MS. About 200 new cases are diagnosed eachweek in the United States. It is an expensive disease to treat, and thedirect and indirect health care costs range from $8,528 to $54,244 perpatient per year in the United States.

Multiple sclerosis disrupts the ability of parts of the nervous systemto communicate, resulting in a range of physical, mental, and sometimespsychiatric problems. There is no known cure for MS, but currenttreatments attempt to improve function after an attack and prevent newattacks. Most medications used to treat MS may be effective inrelapsing-remitting forms of the disease, but generally are ineffectivein progressive forms that are characterized by a chronic demyelinationof axons. Although, the immunomodulator ocrelizumab recently was shownto be effective in the progressive forms, ocrelizumab is associated withmajor potential side effects and poorly tolerated. See Montalban X. etal., “Ocrelizumab versus Placebo in Primary Progressive MultipleSclerosis,” New England Journal of Medicine 376 209-220 (2017).

In another approach, US 2013/0226133 describes a method of restoring themyelin sheath of nerve fibers using stephaglabrin sulfate. In anotherapproach, US 2004/0141947 discloses a method for treating demyelinatingCNS diseases using a colony stimulating factor or colony stimulatingfactor-like ligand such as sargramostim, a type 1 interferon-congener,and at least one additional therapeutic agent. In another approach, US2004/0053850 describes a method of treating a demyelinating disease ofthe CNS by co-administering the tripeptide gly-pro-glu and an AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)/kainate antagonistcompound. In another approach, U.S. Pat. No. 4,760,092 claims a methodof treating demyelinating diseases such as multiple sclerosis usingcolchicine or colchiceine. In another approach, US 2013/0302410describes a method for neuroprotection in demyelinating diseases usingdimethyl fumarate or monomethyl fumarate. In another approach, US2013/0108643 describes a method of treating an autoimmune orinflammatory disease using an inhibitor of macrophage scavenger receptorclass 1 MSR1. In another approach, EP 0423943 describes the use of aninhibitor of a member of the mammalian collagenase family of enzymes totreat demyelinating diseases.

Despite these proposed approaches, there remains a need for methods ofpromoting remyelination in subjects suffering from demyelinatingdiseases, such as MS.

SUMMARY OF THE INVENTION

In accordance with some embodiments, there are provided methods ofpromoting remyelination in a subject in need thereof, comprisingadministering to the subject an effective amount of a steroid hormoneand a Hedgehog signaling pathway modulator. In some embodiments, thesteroid hormone is an androgen receptor ligand such as testosterone, aprogesterone receptor ligand, such as progesterone or allopregnanolone,an estrogen receptor ligands such as estradiol, or isdehydroepiandrosterone, or is a selective hormone receptor modulatorsuch as a selective androgen receptor modulator, a selective estrogenreceptor modulator, or a selective progesterone receptor modulator. Insome embodiments, the Hedgehog signaling pathway modulator is aSmoothened (Smo) agonist, such as3-Chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide(SAG). In some embodiments, the Hedgehog signaling pathway modulator isa Hedgehog signaling pathway antagonist, such as a Gli antagonist. Insome embodiments, the Hedgehog signaling pathway antagonist is2,2′-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine(GANT-61). In some embodiments, wherein the method comprisingadministering both a Smo agonist and a Gli antagonist, such as the Smoagonist is SAG and the Gli antagonist GANT-61.

In some embodiments, the steroid hormone and the Hedgehog signalingpathway modulator are administered in separate compositions,substantially simultaneously or sequentially. In other embodiments, thesteroid hormone and the Hedgehog signaling pathway modulator areadministered in the same composition.

In some embodiments, one or both of the steroid hormone and the Hedgehogsignaling pathway modulator is administered intranasally in anintranasal pharmaceutical composition that further comprises: (a) atleast one lipophilic or partly lipophilic carrier present in an amountof from about 60% to about 98% by weight of the formulation; (b) atleast one compound having surface tension decreasing activity present inan amount of from about 1% to about 20% by weight of the formulation;and (c) at least one viscosity regulating agent present in an amount offrom about 0.5% to about 10% by weight of the formulation. In someembodiments, the intranasal pharmaceutical composition comprises thesteroid hormone. In some embodiments, the intranasal pharmaceuticalcomposition comprises the Hedgehog signaling pathway modulator. In someembodiments, the intranasal pharmaceutical composition comprises thesteroid hormone and the Hedgehog signaling pathway modulator. In someembodiments, the intranasal pharmaceutical composition comprises thesteroid hormone, an Smo agonist, and a Gli antagonist.

In some embodiments, one or both of the steroid hormone and the Hedgehogsignaling pathway modulator is administered intranasally in anintranasal pharmaceutical composition comprising a porous excipient,wherein the steroid hormone and/or the Hedgehog signaling pathwaymodulator is/are loaded onto a surface of the porous excipient locatedinside pores of the porous excipient. In some embodiments, the steroidhormone is loaded onto a surface of the porous excipient located insidepores of the porous excipient. In some embodiments, the Hedgehogsignaling pathway modulator is loaded onto a surface of the porousexcipient located inside pores of the porous excipient. In someembodiments, the steroid hormone and the Hedgehog signaling pathwaymodulator are loaded onto a surface of the porous excipient locatedinside pores of the porous excipient. In some embodiments, the steroidhormone, an Smo agonist, and a Gli antagonist are loaded onto a surfaceof the porous excipient located inside pores of the porous excipient.

In accordance with any embodiments, the subject may be a human, anon-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, amouse, or a rat.

In accordance with any embodiments, the subject may be suffering from ademyelinating disease, such as a demyelinating disease of the centralnervous system selected from multiple sclerosis, amyotrophic lateralsclerosis, Devic's disease, inflammatory demyelinating diseases, centralnervous system neuropathy, central pontine myelinolysis, myelopathies,tabes dorsalis, syphilitic myelopathy, leukoencephalopathies likeprogressive multifocal leukoencephalopathy, Leukodystrophies, andAlzheimer's disease, or a demyelinating disease of the peripheralnervous system selected from Guillain-Barré syndrome, chronicinflammatory demyelinating polyneuropathy, Anti-MAG peripheralneuropathy, Charcot-Marie0Tooth disease, hereditary neuropathy withliability to pressure palsy, peripheral neuropathy, myelopathy, opticneuropathy, and progressive inflammatory neuropathy.

Also provided are a steroid hormone as described herein and a Hedgehogsignaling pathway modulator as described herein for use in a method asdescribed herein for promoting remyelination in a subject in needthereof.

Also provided are uses of a steroid hormone as described herein and/or aHedgehog signaling pathway modulator as described herein in thepreparation of a medicament for treating demyelination in a subject inneed thereof, wherein the method comprises administering the steroidhormone and the Hedgehog signaling pathway modulator to the subject asdescribed herein.

In accordance with other embodiments, there are provided methods ofproducing oligodendroglial cells, comprising culturing primary mixedglial cells in a medium comprising a steroid hormone as described hereinand a Smoothened agonist as described herein.

In accordance with other embodiments, there are provided methods ofdifferentiating oligodendroglial cells into myelin-producing cells,comprising incubating oligodendroglial cells in a medium comprising asteroid hormone as described herein and a Hedgehog signaling pathwayantagonist as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show that Hedgehog and androgen hormone signaling componentsare dynamically transcribed during the last wave of oligodendrogenesisand the myelination process in the early postnatal dorsal telencephalon.FIG. 1A shows the relative expression of transcripts encoding myelinbasic protein (Mbp). FIG. 1B shows the relative expression of theHedgehog signaling Shh ligand. FIG. 1C shows the relative expression ofthe Hedgehog signaling component transcription factor Gli1. FIG. 1Dshows the relative expression of the main receptor mediating androgensignaling (AR). Expression is reported relative to GAPDH, as determinedby quantitative RT-PCR in the dorsal telencephalon from male (grey bars)or female (black bars) mouse pups aged 0, 3, 8 or 15 days. Sexualdimorphism is only detected for AR expression. The values reported arethe mean±SEM from 3 pups per gender per age. *, p≤0.05.

FIGS. 2A-2B show the functional interaction between the Hedgehog andtestosterone signaling pathways in vitro for the control of OPCproliferation and differentiation. FIG. 2A shows the quantification ofOlig2⁺ cells which incorporated the proliferation marker BrdU 2 hoursbefore the end of the culture, and reveals a synergic effect of SAG (0.1μM) and testosterone (1 μM). FIG. 2A also shows that the Smo antagonistSANT-1 (SANT-1, 0.1 μM) blocks testosterone-induced increase in Olig2⁺BrdU⁺ cells. FIG. 2B shows the number of Plp⁺GFP⁺× oligodendroglialcells differentiated from primary mixed glial cells and thus co-expressthe myelin marker Mbp evaluated in the absence (Ctrl) or in the presenceof SAG, SANT-1, or testosterone (T). The values reported are themean±SEM from 3-4 independent cultures. *, p≤0.05; **, p≤0.01; ***,p≤0.001 compared to Ctrl. Comparisons between the different drugs are asindicated: ^(#), p≤0.05; ^(##), p≤0.01; ^(###), p≤0.001; ^($$$),p≤0.001.

FIGS. 3A-3D show that blocking Hedgehog signaling potentiates thedifferentiation of OPCs induced by testosterone in vivo during the lastwave of oligodendrogenesis in the dorsal forebrain. Postnatal day 10male pups were treated with the Smo agonist SAG, the Smo antagonistSANT-1, the steroid hormone testosterone (T), SAG and T, SANT-1 and T,or treated with carrier as control (Ctrl). FIG. 3A shows quantificationof the number of oligodendroglial and astroglial cells in brain slicesof the mice treated as indicated based on immunostaining of thetranscription factor Olig2. FIG. 3B shows quantification of theoligodendrocyte progenitor cells (OPCs) in brain slices of the micetreated as indicated based on immunostaining of the platelet-derivedgrowth factor receptor A (PDGFRα). FIG. 3C shows quantification of themature oligodendrocytes (OLs) in brain slices of the mice treated asindicated based on immunostaining of the adenomatous polyposis coli(APC). FIG. 3D shows the quantification of the myelinating OLsexpressing the myelin basic protein (MBP) in brain slices of the micetreated as indicated. Remarkably, the maturation into MBP⁺ OLs which isrequired for axon wrapping and segment elongation induced bytestosterone is potentiated by the blockade of the Hedgehog signaling.The values reported are the mean±SEM from 3-5 animals per condition. *,p≤0.05; **, p≤0.01; ***, p≤0.001 compared to Ctrl. Comparisons betweenthe different drugs are as indicated: ^(#), p≤0.05.

FIGS. 4A-4E show that the pharmacological activation of Smo enhances thedensity of OPCs/mature OLs and specifically promotes a precocious switchin microglia activation towards the pro-regenerative phenotype in amouse model of demyelination of the central nervous system. FIG. 4Ashows a histogram that visualizes the quantification of PDGFRα+ OPCs perunit surface based on immunostaining of brain slices derived from adultmale mice 10 days after injection of lysolecithin (LPC) into the corpuscallosum in the absence (Ctrl, white bars) or in the presence of SAG(black bars). FIG. 4B shows a histogram that visualizes thequantification of Olig2⁺/APC⁺ mature OLs per unit surface area and thepercentage of Olig2+ oligodendroglial lineage cells which co-express theAPC marker based on immunostaining of brain slices derived from adultmale mice 10 days after injection of lysolecithin (LPC) into the corpuscallosum in the absence (Ctrl, white bars) or in the presence of SAG(black bars). The values reported are the mean±SEM from n=4-6 animalsper condition. FIG. 4C shows histograms quantifying Ki67+/PDGFRα+ OPCsper unit surface area based on immunostaining of brain slices derivedfrom adult male mice 2 days after injection of lysolecithin (LPC) intothe corpus callosum in the absence (Ctrl, white bars) or in the presenceof SAG (black bars). FIG. 4C further shows that a higher number ofproliferating OPCs are observed in the SAG-treated condition based onKi67 and PDGFRα immunostaining (left panel), but the number of PDGFRα+cells are unchanged by SAG treatment (right panel) at this early timepoint. FIG. 4D shows a histogram visualizing quantification of GFAP+astrocytes as percentage of total area. FIG. 4E shows histogramsvisualizing quantification of Iba1+, Arg1+ cells per surface unit (leftpanel), and Arg1+ cells as percentage of Iba1+ cells (right panel) basedon immunostaining of brain slices derived from adult male mice 2 daysafter injection of lysolecithin (LPC) into the corpus callosum in theabsence (Ctrl, white bars) or in the presence of SAG (black bars). FIG.4E further shows that a much higher number of Arg-1⁺ pro-regenerativemicroglia is detected in the lesion of SAG-treated animals.

FIGS. 5A-5F show that combination therapy based on the simultaneouspharmacological activation of Smo-mediated Hh signaling and androgensignaling highly mitigates the course of experimental autoimmuneencephalomyelitis (EAE). FIG. 5A shows EAE clinical scores aftertherapeutic administration of SAG and testosterone used separately orsimultaneously compared with vehicle administration. FIG. 5B showselectron micrographs from the lumbar spinal cord of vehicle (Ctrl),testosterone (T), SAG and SAG+T-treated EAE mice. Besides normalmyelinated axons (right-most arrow), demyclinated (bottom-most arrow)and abnormal (left-most arrow) axons are observed at a higher level inthe control condition. FIG. 5C shows an analysis of the g-ratio (axondiameter/axon+myelin diameter) and indicates that the values aresignificantly lower when testosterone and SAG are used separately orsimultaneously as compared to the control. SAG alone or withtestosterone displays a significantly higher effect on the g-ratio thantestosterone alone. FIG. 5D shows the quantification of abnormal axons,including axons exhibiting myelin still compacted but detached from theaxon, double myelin sheaths or multilayered myelin where the inside ofthe axon was obstructed. The percentage of abnormalities among the totalnumber of axons is significantly decreased under treatment conditionscompared to the control. FIG. 5F-5F show the quantification of the areaoccupied by Iba1⁺ (FIG. 5E) and Arg1⁺ (FIG. 5F) cells expressed as apercentage of the total area of the lesion in the image based onimmunostaining of Iba and Arg1 of the lumbar spinal cord of EAE animalstreated by the vehicle (Ctrl) or by the drugs testosterone and SAG usedseparately or simultaneously. FIG. 5G shows the quantification of theGFAP-positive area as the percentage of the lesion area in the spinalcord of the EAE animals treated with testosterone and SAG, separately orsimultaneously. FIG. 5H shows the quantification of the Claudin-positivearea as the percentage of the lesion area in the EAE animals treatedwith testosterone and SAG, separately or simultaneously. Data reportedare the mean±SEM (n=10 mice per condition). *, p≤0.05, **, p≤0.01, ***,p≤0.001, ^(##), p=0.001, **** p<0.0001, one-way ANOVA with Tukey'smultiple comparison test.

FIG. 6 shows that testosterone (T) and estradiol (E2), itsaromatase-mediated conversion product whose generation is prevented bythe presence of the aromatase inhibitor fadrozole (Fad), both regulatethe expression of phosphorylated STAT3 in astrocytes. The valuesreported are the means±SEM from n=4 animals. *, p≤0.05; **, p≤0.01.

DETAILED DESCRIPTION

Described herein are methods of promoting remyelination in a subject inneed thereof that comprise administering to the subject an effectiveamount of a steroid hormone and a Hedgehog signaling pathway modulator.In some embodiments, the methods are for treating a demyelinatingdisease, such as MS. Also described are related compositions and uses ofa steroid hormone and a Hedgehog signaling pathway modulator. Furtherdescribed are compositions and methods using a steroid hormone, aSmoothened agonist, and a Hedgehog signaling pathway antagonist. Alsodescribed are in vitro methods of producing proliferatingoligodendroglial cells that involve culturing primary mixed glial cellsin a medium comprising a steroid hormone and a Smo agonist. Alsodescribed are in vitro methods of differentiating oligodendroglial cellsinto myelin-producing cells that involve incubating oligodendroglialcells in a medium comprising a steroid hormone and an Smo antagonist.

Definitions

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Materials, reagents andthe like to which reference is made in the following description andexamples are obtainable from commercial sources, unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only.

The term “about” means that the number comprehended is not limited tothe exact number set forth herein, and is intended to refer to numberssubstantially around the recited number while not departing from thescope of the invention. As used herein, “about” will be understood bypersons of ordinary skill in the art and will vary to some extent on thecontext in which it is used. If there are uses of the term which are notclear to persons of ordinary skill in the art given the context in whichit is used, “about” will mean up to plus or minus 10% of the particularterm.

As used herein, “Smoothened” or “Smo” is a 7-transmembrane GPCR-likereceptor that is primarily located in the membrane of intracellularvesicles or at the plasma membrane when it is activated. Smo is acomponent of the Sonic Hedgehog (Shh) signaling pathway. The Shh pathwayacts to control oligodendrocyte generation during embryonic development.See e.g. Traiffort E. et al., “Hedgehog: A key signaling in thedevelopment of the oligodendrocyte lineage,” Dev. Biol. 4:28 (2016);Ferent and Traiffort, “Hedgehog: Multiple Paths for Multiple Roles inShaping the Brain and Spinal Cord,” Neuroscientist 21:356-71 (2015).

As used herein, “subject” denotes any mammal in need of treatment for ademyelinating disease or condition or in need of promotion ofremyelination, including humans. For example, a subject may be sufferingfrom or at risk of developing a demyelinating disease or condition.

As used herein, the term “administering” includes directly administeringto another, self-administering, and prescribing or directing theadministration of an agent as disclosed herein.

As used herein, the phrases “effective amount” and “therapeuticallyeffective amount” mean that active agent dosage or plasma concentrationin a subject, respectively, that provides the specific pharmacologicaleffect for which the active agent is administered in a subject in needof such treatment. It is emphasized that an effective amount of anactive agent will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be an effective amount by those of skill in the art.

As used herein, the term “pharmaceutical composition” refers to one ormore active agents formulated with a pharmaceutically acceptablecarrier, excipient or diluent.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in vivowithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Methods of Promoting Remyelination

The methods described herein are based on the surprising discovery thattreatment with a steroid hormone and Hedgehog signaling pathwaymodulator dramatically increases the number of oligodendrocytes andmyelin-producing cells. Although therapies using only a steroid hormoneor only an Hedgehog signaling pathway modulator have been described(see, e.g., E1-Etr et al., “Hormonal influences in multiple sclerosis:new therapeutic benefits for steroids,” Maturitas 68:47-51 (2011);Bielecki et al., “Unexpected central role of the androgen receptor inthe spontaneous regeneration of myelin,” Proc. Natl. Acad. Sci.113:14829-14834 (2016); Samanta et al., “Inhibition of Gli1 mobilizesendogenous neural stem cells for remyelination,” Nature 15:448-52(2015); US 2015/0011610; Ferent et al., “Sonic Hedgehog signaling is apositive oligodendrocyte regulator during demyelination,” J.Neuroscience 33:1759-72 (2013)), the present inventors found that usinga steroid hormone together with a Hedgehog signaling pathway modulatorsynergistically enhances production of oligodendrocytes and myelinproducing cells, leading to improved promotion of remyelination andoffering more effective treatments for demyelinating diseases.

In this regard, the present inventors discovered an overlappingexpression pattern of Shh signaling components and the androgen receptorduring early development of oligodendrocytes. See Example 1, FIG. 1.These expression patterns appeared to be consistent with the firstdemonstration of a functional interaction between Shh signaling andsteroid hormones during the myelination process. Indeed, the inventorsdiscovered that treatment with an androgen (such as testosterone) canpromote the proliferation of oligodendrocyte precursor cells at a higherlevel in the presence of an Smo agonist as compared to in the absence ofan Smo agonist. In addition, it was found that the concomitant use of anSmo antagonist and testosterone promotes the differentiation of myelinproducing cells in a synergistic manner. See Example 1, FIG. 2, andExample 1, FIG. 3. These results support the methods described herein.

The present inventors also surprisingly discovered that, in the contextof myelin repair, a Smo agonist promotes a precocious switch inmicroglia activation towards the pro-regenerative phenotype and can actsynergistically with a steroid hormone (such as testosterone) to promoteremyelination, as shown in the examples reported below. These resultsalso support the methods described herein.

Steroid Hormones

Steroid hormones useful in the compositions and methods described hereininclude but are not limited to steroid hormones of the progestogen,estrogen, and androgen families, synthetic steroid hormones, andselective hormone receptor modulators.

In some embodiments, the steroid hormone is an androgen receptor ligand(e.g., an androgen). Androgens are a group of steroid hormones thatmediate their effects through binding and activation of the androgenreceptor (AR). As used herein, androgens include testosterone anddihydrotestosterone. In specific embodiments, the steroid hormone is theandrogen testosterone.

In some embodiments, the steroid hormone is a progestogen. Progestogensare a group of steroid hormones that mediate their effects throughbinding and activation of the progesterone receptor (PR). In somespecific embodiments, the progestogen is progesterone orallopregnanolone, which is derived from progesterone and activates theγ-aminobutyric acid (GABA) receptor.

In some embodiments, the steroid hormone is an estrogen receptor ligand(e.g., an estrogen). Estrogens are a group of steroid hormones thatmediate their effects through binding and activation of the estrogenreceptor (ER). As used herein, estrogens include estradiol.

In some embodiments, the steroid hormone is dehydroepiandrosterone(DHEA). DHEA can serve as precursor for both androgenic and estrogenicsteroids.

In some embodiments, the steroid hormone is a synthetic steroid.

In some embodiments, a selective hormone receptor modulator is used as a“steroid hormone” in the methods described herein. Selective hormonereceptor modulators function similarly to steroid hormone but generallyare more selective than steroids per se. As used herein, “selectivehormone receptor modulators” include, but are not limited to, selectiveandrogen receptor modulators, selective estrogen receptor modulators,and selective progesterone receptor modulators.

Hedgehog Signaling Pathway Modulators

Hedgehog signaling pathway modulators useful in the compositions andmethods described herein include Smo agonists that increase Shhsignaling and Smo antagonists that decrease Shh signaling, as well asGli antagonists.

Thus, in some embodiments, the Hedgehog signaling pathway modulator isan Smoothened (Smo) agonist. Smo agonists may interact with Smoreceptors to activate the downstream Gli transcription factor. See,e.g., Hadden et al., “Hedgehog Pathway Agonism: Therapeutic Potentialand Small-Molecule Development.” Chem. Med. Chem. 9:27-37 (2014); Chenet al., “Small molecule modulation of Smoothened activity,” Proc. Natl.Acad. Sci. 99:14071-14076 (2002). Examples of Smoothened agonistsinclude3-Chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide(SAG),3-chloro-4,7-difluoro-N-(4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide(Hh Ag-1.5), glucocorticoids, 9H-Purin-6-amine,9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)(Purmorphamine), propyl4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate(GSA-10), cholesterol, and osteogenic (1H)-quinolone-based compoundssuch as GSA-10 like compounds 20 and 25 (Manetti et al., “Design,synthesis and biological characterization of a new class of osteogenic(1H)-quinolone derivatives,” Eur. J. Med. Chem. 121:747-757 (2016)). Inspecific embodiments, the Hedgehog signaling pathway modulator is theSmo agonist3-Chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide(SAG).

In some embodiments, the Hedgehog signaling pathway modulator is a Gliantagonist. Gli antagonists include but are not limited to, smallmolecule Gli1 antagonists such as2,2′-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethyl-benzenamine(GANT61) and 2,3,4,5-Tetra(4-pyridyl)thiophene,4,4′,4″,4″′-Thiene-2,3,4,5-tetrayltetrapyridine (GANT58) disclosed inLauth et al., “Inhibition of GLI-mediated transcription and tumor cellgrowth by small-molecule antagonists,” Proc. Natl. Acad. Sci. 104:8455-60 (2007). Both GANT61 and GANT58 are believed to act in thenucleus to block Gli function, and GANT61 is believed to interfere withDNA binding of Gli1. In specific embodiments, the Hedgehog modulator isthe Gli antagonist GANT61.

Other Hedgehog signaling pathway modulators are Smo antagonists, such asdisclosed in Chen (2002) (supra) and Rimkus et al., “Targeting the SonicHedgehog signaling pathway: Review of Smoothened and Gli inhibitors,”Cancers 8: pii:E22 (2016), including(4-Benzyl-piperazin-1-yl)-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-ylmethylene)-amine(SANT-1),N-[3-(1H-Benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-triethoxybenzamideSANT-2, and(4-Benzyl-piperazin-1-yl)-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-ylmethylene)-amine(SANT-3), and SANT-4, which has the following structure:

In some embodiments, both an Smo agonist and a Gli antagonist are usedas Hedgehog signaling pathway modulators. That is, in some embodiments,the Hedgehog signaling pathway modulator includes both an Smo agonistand a Gli antagonist. As such, in some embodiments, a steroid hormone,an Smo agonist and a Gli antagonist are used or administered asdescribed herein.

Pharmaceutical Compositions

The steroid hormone and Hedgehog signaling pathway modulator can beadministered in separate compositions (substantially simultaneously orsequentially), or they can be administered in the same composition.

The composition(s) can be any pharmaceutical compositions suitable foradministering the steroid hormone and/or Hedgehog signaling pathwaymodulator, formulated for any route of administration. Suitable routesof administration may, for example, include oral, rectal, transmucosal,especially intranasal, intestinal or parenteral delivery, includingintramuscular, subcutaneous and intramedullary injections as well asintrathecal, direct intraventricular, intracardiac, e.g., into the rightor left ventricular cavity, into the common coronary artery,intravenous, intraperitoneal, intranasal, or intraocular injections.Some embodiments involve oral administration. Some embodiments involveintranasal administration.

In some embodiments, the steroid hormone and/or Hedgehog signalingpathway modulator is formulated in an intranasal pharmaceuticalcomposition. As used herein “intranasal composition” means a compositionsuitable for, or adapted for, intranasal delivery. Such embodiments mayoffer enhanced uptake of the steroid hormone and/or Hedgehog signalingpathway modulator.

Exemplary oleogel-type intranasal pharmaceutical compositions fortestosterone have been described, for example, in U.S. Pat. No.8,574,622, the entire contents of which are incorporated herein byreference. In some embodiments, one or both of the steroid hormone andthe Hedgehog signaling pathway is formulated in an intranasalpharmaceutical composition as described in U.S. Pat. No. 8,574,622, suchas a composition that includes the active agent(s) and that furthercomprises: (a) at least one lipophilic or partly lipophilic carrierpresent in an amount of from about 60% to about 98% by weight of theformulation; (b) at least one compound having surface tension decreasingactivity present in an amount of from about 1% to about 20% by weight ofthe formulation; and (c) at least one viscosity regulating agent presentin an amount of from about 0.5% to about 10% by weight of theformulation.

In such oleogel embodiments, the lipophilic or partly lipophilic carriermay be any such carrier suitable as a carrier or vehicle for a nasalpharmaceutical composition, such as an oil, such as a vegetable oil,such as castor oil, hydrogenated castor oil, soybean oil, sesame oil, orpeanut oil, or any vehicle discussed below that is lipophilic or partlylipophilic, or any other suitable lipophilic or partly lipophiliccarrier.

In such oleogel embodiments, the compound(s) having surface tensiondecreasing activity may be one or more surfactants such as lecithin,fatty acid esters of polyvalent alcohols, of sorbitanes, ofpolyoxyethylensorbitans, of polyoxyethylene, of sucrose, of polyglyceroland/or one or more humectants such as sorbitol, glycerine, polyethyleneglycol, and macrogol glycerol fatty acid esters, or one or more oleoylmacrogolglycerides (such as LABRAFIL® M 1944 CS, available fromGattefosse (France), or any surfactant discussed below, or any othersuitable surfactant.

In such oleogel embodiments, the viscosity regulating agent(s) may beone or more selected from thickeners and gelling agents, such ascellulose and cellulose derivatives, polysaccharides, carbomers,polyvinyl alcohols, povidone, colloidal silicon dioxide, cetyl alcohols,stearic acid, beeswax, petrolatum, triglycerides and lanolin, or anyviscosity regulating agent discussed below, or any other suitablesurfactant.

Other exemplary intranasal pharmaceutical compositions include thosedescribed in U.S. patent application Ser. No. 15/612,454, the entirecontents of which are incorporated herein by reference. U.S. patentapplication Ser. No. 15/612,454 describes intranasal pharmaceuticalcompositions wherein an active agent is loaded onto a porous agent.Thus, in some embodiments, one or both of the steroid hormone and theHedgehog signaling pathway modulator is formulated in an intranasalpharmaceutical composition as described in U.S. patent application Ser.No. 15/612,454, such as a composition comprising a porous agent, whereinthe steroid hormone and/or the Hedgehog signaling pathway modulatoris/are loaded onto a surface of the porous agent located inside pores ofthe porous agent. As described in U.S. Ser. No. 15/612,454, theactive-agent loaded porous agent may itself be formulated in an oleogelcomposition, such as described those in U.S. Pat. No. 8,574,622.

In such porous agent embodiments, the porous agent may comprise aninorganic porous material, such as colloidal silicon dioxide,micro-porous silicon dioxide, meso-porous silicon dioxide, macro-poroussilicon dioxide, polyorganosiloxanes, pharmaceutical clays, silicondioxide nanotubes, silicon dioxide gel, magnesium alumosilicate (such asbut not limited to VEEGUM® from Vanderbilt Minerals, LLC), activatedcarbon, anhydrous calcium phosphate, calcium carbonate, alumina, andcombinations of any two or more thereof. Exemplary inorganic porousmaterials include porous silicon dioxide commercially available underthe SYLOID® brand from W.R. Grace & Co. (such as but not limited toSYLOID® 244FP, 72FP, XDP6035 (also known as SILSOL™ 6035), XDP3050,XDP3150, AL-1FP, and combinations of any two or more thereof), poroussilicon dioxide available under the AEROPERL® brand from EvonikIndustries, Corp. (such as but not limited to AEROPERL® 300, which has asurface area of about 260 to 320 m²/g (such as about 300 m²/g), a porevolume of about 1.5 to 1.9 ml/g, and an average particle size of about20 to about 60 μm), silicon dioxide PARTECK® SLC from EMD Millipore,NEUSILIN® (a synthetic, amorphous form of magnesium aluminometasilicate)from Fuji Chemical Industry, Zeolite Socony Mobil-5, Mobil Compositionof Matter No. 41, SBA-15, FDU-11, OMS-7, OMS-Lemon-7, and II™-56. Insome embodiments, the porous agent comprises silicon-based powders,which may be hydrophobic or hydrophilic, e.g., depending on groupschemically bonded to their surfaces.

In some embodiments, the porous agent comprises an organic-inorganichybrid, such as metal-organic frameworks (MOFs). Exemplary hybridmaterials can be formed by self-assembly of polydentate bridging ligandsand metal connecting points.

In some embodiments, the porous agent comprises organic polymers, suchas microporous organic polymers, polystyrene, cellulose, and/orpoly(methyl methacrylate). In some embodiments, microporous organicpolymers are formed by carbon-carbon coupling reactions and comprised ofnon-metallic elements such as carbon, hydrogen, oxygen, nitrogen, and/orboron. In some embodiments, organic polymers are produced by emulsionpolymerization and hypercrosslinking followed by chemical etching ofsacrificial SiO₂ cores. In some embodiments, networks of organicpolymers are constructed from small organic building blocks.

In some embodiments, the porous agent comprises porous materials basedon complexing agents, such as an ion exchange resin (such as but notlimited to cross-linked polystyrene) or an adsorbent (such as but notlimited to β-cyclodextrin-based porous silica, α-cyclodextrin-basedporous silica, hydroxpropyl-β-cyclodextrin-based porous silica, andporous materials based on other adsorbent resins).

In some embodiments, the surface of the porous agent—including the innerpore surface—is functionalized to bind the active agent(s) and/orcontrol release of the active agent(s) after a certain amount of time orin response to a stimulus.

The active agent-loaded porous agent may be formulated in any vehiclesuitable as a vehicle for a nasal pharmaceutical composition. In someembodiments, the vehicle for the porous agent is a hydrophilic vehicle.In some embodiments, the vehicle is a lipophilic or partly lipophilicvehicle, such as a vehicle comprising one or more fats, oils, waxes,phospholipids, steroids (e.g., cholesterol), sphingolipids, ceramides,sphingosines, prostaglandins, and/or fat-oil vitamins. In someembodiments, the vehicle comprises an oil or a mixture of oils, such asvegetable oil, castor oil, hydrogenated castor oil, soybean oil, sesameoil, or peanut oil; fatty acid esters, such as ethyl- and oleyl-oleate,isopropylmyristate; medium chain triglycerides; glycerol esters of fattyacids; polyethylene glycol; phospholipids; white soft paraffin; orcombinations of any two or more thereof.

The vehicle may be present in any suitable amount, such as an amounteffective to provide desired properties for nasal administration,desired physical properties, desired release properties, desiredpharmacokinetics, etc. In some embodiments, the composition comprises avehicle in an amount of from about 15% to about 98% by weight, about 30to about 98% by weight, about 50% to about 95% by weight, about 75% toabout 95% by weight, about 80%, or about 90% by weight, based on thetotal weight of the composition. In some embodiments, the compositioncomprises a vehicle in an amount of from 15% to 98% by weight, 30 to 98%by weight, 50% to 95% by weight, 75% to 95% by weight, 80%, or 90% byweight, based on the total weight of the composition.

The active agent-loaded porous agent may be formulated with one or morecompounds having surface decreasing activity, e.g., surfactants. Thesurfactant, if present, may be any surfactant suitable for use as asurfactant in a nasal pharmaceutical composition. In some embodiments,the surfactant is selected from anionic, cationic, amphoteric, andnon-ionic surfactants, including, but not limited to, lecithin, fattyacid esters of polyvalent alcohols, fatty acid esters of sorbitanes,fatty acid esters of polyoxyethylensorbitans, fatty acid esters ofpolyoxyethylene, fatty acid esters of sucrose, fatty acid esters ofpolyglycerol, oleoyl polyoxylglycerides (such as but not limited toapricot kernel oil PEG-6-esters), oleoyl macrogolglycerides, and/orhumectants such as sorbitol, glycerine, polyethylene glycol, macrogolglycerol fatty acid ester, and combinations of any two or more thereof.In some embodiments, the surfactant comprises an oleoylmacrogolglyceride (such as LABRAFIL® M 1944 CS (Gattefosse,Saint-Priest, France)) or a mixture of olcoyl macrogolglycerides.

The active agent-loaded porous agent may be formulated with one or moreviscosity-regulating agents, which may be any viscosity-regulating agentsuitable for use as a viscosity-regulating agent in a nasalpharmaceutical composition. In some embodiments, theviscosity-regulating agent comprises mesoporous silica (which may beloaded with active agent or unloaded). In some embodiments, theviscosity-regulating agent comprises cellulose, cellulose-containingsubstances, polysaccharides, carbomers, polyvinyl alcohol, povidone,colloidal silicon dioxide, cetyl alcohols, stearic acid, beeswax,petrolatum, triglycerides, lanolin, or combinations of any two or morethereof. In some embodiments, the viscosity-regulating agent comprisescolloidal silicon dioxide (such as but not limited to AEROSIL® 200(Evonik) and/or CAB-O-SIL® M5 (Cabot)). In some embodiments, theviscosity-regulating agent comprises synthetic silica, such as SYLODENT®(precipitated silica with a compacted bulk density of about 110 kg/m³, aspecific surface area of about 190 m²/g, and an average particle size ofabout 18 μm) or SYLOBLANC® silicas (porous silica gel with a pore volumeof about 1.6 mug and an average particle size of about 3 μm) from W.R.Grace & Co. In some embodiments, the viscosity-regulating agentcomprises hydrophilic fumed silica, such as AEROSIL® 200 and/orlipophilic silicon dioxide, such as AEROSIL® R972 (which is fumed silicaafter-treated with dimethyldichlorosilane, and which has a surface areaof about 90 to about 130 m²/g). Without being bound by theory, it isbelieved that hydrophilic fumed silica can be used to prepare athixotropic gel composition with a high temperature stability ascompared to a comparable gel produced with other viscosity-regulatingagents.

The viscosity-regulating agent, if present, may be present in an amounteffective to adjust the viscosity of the composition to the desiredlevel. In some embodiments, the composition comprises from about 0.5 toabout 20% by weight, about 0.5 to about 10% by weight, about 0.5 toabout 7% by weight, about 1 to about 4% by weight, about 4% by weight,or about 2% by weight viscosity-regulating agent, based on the totalweight of the composition. In some embodiments, the compositioncomprises from 0.5 to 20% by weight, 0.5 to 10% by weight, 0.5 to 7% byweight, 1 to 4% by weight, 4% by weight, or 2% by weightviscosity-regulating agent, based on the total weight of thecomposition.

Regardless of the specific formulation used, the steroid hormone andHedgehog signaling pathway modulator are formulated to provide atherapeutically effective amount of the active agents in doses suitablefor the route of administration, such as a volume of compositionsuitable for administration to one or both nostrils, a volume suitablefor oral administration, or a volume suitable for intravenous,subcutaneous, or intramuscular administration.

Methods of Use

As noted above, in accordance with the methods and uses describedherein, the steroid hormone and Hedgehog signaling pathway modulator areadministered to a subject in need thereof, such as a subject in need ofpromotion of remyelinaion and/or a subject in need of treatment for ademyelinating disease or condition. The subject may be any mammal, suchas a human, non-human primate, dog, cat, cow, sheep, horse, rabbit,mouse, or rat.

Demyelinating diseases can be divided in those affecting the centralnervous system and those affecting in the peripheral nervous system,which present different demyelination conditions. In some embodiments,the subject is suffering from a demyelination disease of the CNS such asmultiple sclerosis, amyotrophic lateral sclerosis, Devic's disease,inflammatory demyelinating diseases, central nervous system neuropathy,central pontine myelinolysis, myelopathies, tabes dorsalis, syphiliticmyelopathy, leukoencephalopathies (including progressive multifocalleukoencephalopathy), leukodystrophies, and Alzheimer's disease. In someembodiments, the subject is suffering from a demyelination disease ofthe peripheral nervous system such as Guillain-Barré syndrome, chronicinflammatory demyelinating polyneuropathy, anti-Myelin AssociatedGlycoprotein peripheral neuropathy, Charcot-Marie-Tooth disease,hereditary neuropathy with liability to pressure palsy, peripheralneuropathy, myelopathy, optic neuropathy, and progressive inflammatoryneuropathy.

As noted above, the steroid hormone and Hedgehog signaling pathwaymodulator can be administered in separate compositions (substantiallysimultaneously or sequentially in any order), or can be administered inthe same composition. As also noted above, in any embodiments, thesteroid hormone and Hedgehog signaling pathway modulator can beadministered by any suitable route of administration, includingintranasal, oral, intravenous, subcutaneous and intramuscular. When thesteroid hormone and Hedgehog signaling pathway modulator areadministered in different compositions, they can be administered by thesame or different routes of administration, such as oral or intranasal.In specific embodiments, the steroid hormone is administered orally orintranasally. Independently, in specific embodiments, the Hedgehogsignaling pathway modulator is administered orally or intranasally.

As also noted above, the steroid hormone and Hedgehog signaling pathwaymodulators are administered in amounts effective to promoteremyelination. As used herein, the term “remyelination” refers to thegeneration of new myelin sheaths. Remyelination can be assessed bymethods such as direct determination of the state of myelin in thesubject, such as by measuring white matter mass using magnetic resonanceimaging (MRI), measuring the thickness of myelin fibers using a magneticresonance spectroscopy (MRS) brain scan, or any other direct measures(e.g., Positron-Emission Tomography (PET), Diffusion-Weighted Imaging(DW-I, or DW-MRI), Diffusion Tensor Imaging, Myelography, MagnetizationTransfer, etc.). Additionally or alternatively, remyelination can beassessed by detecting a reduction in the size or number of inflammatorylesions (i.e., scleroses) present in the patient, monitoring a patient'scerebrospinal fluid (which may be obtain, e.g., by lumbar puncture) fora reduction in the presence or amount of, e.g., (i) abnormal proteinssuch as tiny fragments of myelin, (ii) elevated levels of or specifictypes of lymphocytes, and/or (iii) abnormal levels of immunoglobulin(IgG) molecules; monitoring a patient for a positive change inneuropsychology (e.g., the status of various abilities such as memory,arithmetic, attention, judgment and reasoning); and/or monitoring apatient's urine for a decrease in levels of myelin basic protein-likematerial (MBPLM). Any one or more of these methodologies can be used toassess remyelination, or an alternative method can be used. The methodsdescribed herein are not limited by these or other specificmethodologies for assessing remyelination.

In some embodiments, the steroid hormone is testosterone and isadministered at a dose of from about 0.05 to about 0.5 mg/day, includingfrom about 0.1 to about 0.3 mg/day, including about 0.2 mg/day. Inembodiments using a different androgen receptor ligand, a correspondingmolar amount of the androgen receptor ligand can be used.

In some embodiments, the Hedgehog signaling pathway modulator is SAG,and is administered at a dose of from about 5 to about 25 mg/kg bodyweight of the subject, including from about 10 to about 20 mg/kg,including 15 mg/kg. In embodiments using a different Smo agonist, acorresponding molar amount of Smo agonist can be used.

In some embodiments, the Hedgehog signaling pathway modulator isGANT-61, and is administered at a dose of from about 5 to about 25 mg/kgbody weight of the subject, including from about 10 to about 20 mg/kg,including about 15 mg/kg. In embodiments using a different Hedgehogsignaling pathway antagonist, a corresponding molar amount of Hedgehogsignaling pathway antagonist can be used.

In some embodiments, the amounts of one or both of the steroid hormoneand Hedgehog signaling pathway modulators administered is effective topotentiate the remyelination-promoting activity of the other. Thus, insome embodiments, the amount of steroid hormone administered with agiven amount of Hedgehog signaling pathway modulator is more effectivein promoting remyelination than the same amount of Hedgehog signalingpathway modulator alone. Additionally or alternatively, in someembodiments, the amount of Hedgehog signaling pathway modulatoradministered with a given amount of steroid hormone is more effective inpromoting remyelination than the same amount of steroid hormone alone.

Also provided are steroid hormone and an Hedgehog signaling pathwaymodulator as described herein, for use in a method of promotingremyelination in a subject in need thereof, or for treating ademyelinating disease or condition, as discussed above.

Also provided are uses of a steroid hormone and an Hedgehog signalingpathway modulator as described herein, in the preparation of amedicament as described herein, for use in a method of promotingremyelination in a subject in need thereof, or for treating ademyelinating disease or condition, as discussed above.

As noted above and shown in the examples, the present inventors foundthat the use of a steroid hormone (such as testosterone) and a Smoagonist (such as SAG) synergistically promotes the production ofoligodendrocytes and mitigates the course of autoimmuneencephalomyelitis in experimental models. Furthermore, the use of asteroid hormone (such as testosterone) and a Gli antagonist (such asGANT61) may synergistically promote the production of myelin producingcells.

The following examples are provided to illustrate the invention, but itshould be understood that the invention is not limited to the specificconditions or details of these examples.

EXAMPLES Materials and Methods

Handling of Animals.

Wild-type gonadally intact or castrated C57B1/6 male mice were purchasedat the age of 8 to 12 weeks from Janvier Labs Breeding Center (France).For in vitro experiments, litters were obtained from timely matedC57B1/6 females purchased from Janvier Labs Breeding Center or werein-house bred and mated with Plp-EGFP mice obtained from Dr. WendyMacklin (University of Colorado, USA). See Mallon et al., J.Neuroscience 22: 876-885 (2002). All animals were housed under standardconditions, including a 12 hour light-dark cycle with food and water adlibitum. All procedures were performed according to the EuropeanCommunities Council Directive (86/806/EEC) for the care and use oflaboratory animals and were approved by the Regional Ethics CommitteeCEEA26, Ministère de l'Education Nationale, de l'Enseignement et de laRecherche.

Preparing Active Agent Formulations.

The Smo agonist (SAG) and antagonist (SANT) used were those described inChen et al., “Small molecule modulation of Smoothened activity,” Proc.Natl. Acad. Sci. 99:14071-14076 (2002), purchased from D&C Chemicals(China). (SAG, product number: DC-8225; SANT-1, product number DC-8327)The active agents were dissolved in dimethyl sulfoxide (10 mM) andsubsequently diluted either in culture medium or in 0.9% NaCl to reachthe appropriate concentrations. Testosterone was provided bySigma-Aldrich (France). Testosterone was dissolved in sesame oil (1mg/ml) and then diluted to obtain the desired steroid hormoneconcentrations.

Immunostaining Experiments.

The primary antibodies used for immunostaining were as follows:Oligodendcyte Transcription Factor 2 (Olig2) (Rabbit, Millipore; mouse,Millipore), myelin basic protein (MBP), (Rabbit, Millipore); anti-NG2(rabbit, Millipore); Adenomatus Polyposis Coli (APC/CC1) (mouse,Calbiochem), BrdU antibody (rat, Abcam); Glial Fibrillary Acidic Protein(GFAP), (Rabbit, Dako; mouse, Sigma); Ionized calcium binding adaptormolecule 1 (Iba1, rabbit, Wako); Arginase-1 (goat, Santa-Cruz),Protolipid Protein (PLP), (mouse, Millipore); Platelet-Derived GrowthFactor Receptor alpha (PDGFRa), (mouse, Millipore); Neurofilament 200(NF200), (chicken, Neuromics); Ki67 (mouse monoclonal; BD Pharmingen).The secondary antibodies used were: goat anti-rabbit cyanine 3conjugated (Jackson Immunoresearch); goat anti-mouse Alexa 488,anti-rabbit Alexa 633, anti-chicken Alexa 546; donkey anti-goat Alexa546 (Thermo Fisher Scientific).

Image Acquisition and Analysis.

Images were taken using the microscope analyzing system Axiovision 4.2(Carl Zeiss, Inc.), the confocal Zeiss LSM 510-Meta Confocor 2 and thescanner imager with the CaseViewer software. Analyses were performedwith ImageJ software. At least 10 sections per mouse were analyzed anddata are the mean of 3-5 mice. For the brains derived from thelysolecithin (LPC)-injected animals, immunofluorescent-positive cells orareas were determined in one every other 5 sections throughout the wholedemyelinated lesion per mouse and averaged for each animal. The lesionsurface was determined by measuring the area of the nucleardensification (correlated with myelin loss visualized by MBP or PLPstaining) one every other 5 slices through the whole demyelinatedlesion.

Electron Microscopy.

Ultrathin sections of lumbar spinal cords were examined usingtransmission electron microscope (1011 JEOL) equipped with a Gatandigital camera. The g ratio (the ratio between the axon diameter andfiber diameter corresponding to myelin sheath+axon diameter) wasestimated by measuring the minimum and maximum axon diameter and fiberdiameter for each axon using ImageJ software. At least 300 randomlychosen myelinated axons were evaluated for each animal.

Rt-Qpcr Analysis.

At least four animals of each gender and age were sacrificed bydecapitation. Dorsal telencephalons were dissected and frozen in liquidnitrogen for further processing. Total RNA was isolated by using theTrizol Technique (Thermo Fisher Scientific) and RNeasy Mini Kit(Qiagen). Reverse Transcription was performed using the High CapacitycDNA Reverse Transcription kit (Applied Biosystems). Quantitativereal-time PCR was carried out by using the TaqMan Gene expression MasterMix (Thermo Fisher Scientific) and gene expression was analyzed with the7300 Systems SDS Software (Applied Biosystems) normalized to referencegenes GAPDH. TaqMan probes were as follows: GAPDH, Mm99999915_m1; MBP,Mm01266402_m1; Shh, Mm00436528_m1; Gli1, Mm00494654; AR, Mm00442688.

Statistical Analysis.

Data are expressed as means±S.E.M. Statistical analysis was performedwith GraphPad Prism 6.0. One way ANOVA was used for statisticalsignificance evaluation. The levels of significance were *p<0.05,**p<0.01, ***p<0.001.

Example 1 Functional Interaction Between Hedgehog and Androgen SignalingDuring Early Postnatal Myelination

The present inventors identified a functional interaction betweenHedgehog (Hh) and androgen signaling during the early period ofpostnatal myelination of the telencephalon. In this regard, theexpression profiles of transcripts encoding myelin basic protein (Mbp),Hh signaling components (Shh, Gli1), and the androgen receptor (AR) werestudied, using the dorsal forebrain of male and female mice from birthto postnatal day 15 (P15). This period encompasses the neonatal wave ofoligodendrogenesis, the maturation of the generated oligodendrocyteprecursor cells (OLPs) and the physiological process of myelination inthe dorsal forebrain. Kessaris et al., Nature Neuroscience 9:173-179(2006). Mbp transcription was detected at a very low level at P3 andthen displayed an approximately 10- and 60-fold increase at P8 and P15,respectively, as shown in FIG. 1A. Shh mRNA increased slightly butsignificantly until P8 before reaching a plateau, as shown in FIG. 1B.Unexpectedly, Gli1 progressively decreased between P0 and P15, as shownin FIG. 1C. In contrast, androgen receptor (AR) transcription wasdetected at a low level at birth, but sharply increased until P15,reaching a level 10- to 24-fold higher in males and females,respectively, as shown in FIG. 1D. The transcription of Shh, Gli1 andMbp was not significantly different according to the sex of the animalsat the studied time points. In contrast, although the expression of ARwas significantly higher in males compared to females at birth, AR wastranscribed in a comparable manner regardless of the sex of the animalsat P3 and P8, and AR expression was unexpectedly displayed a slightlybut significantly higher level in females compared to males at P15, asshown in FIG. 1D. These results indicate that the androgen and Hedgehogsignaling pathways might communicate to regulate the myelinationprocess.

SAG+T Promotes Proliferating Oligodendrocytes During DevelopmentalMyelination

SANT+T Promotes Differentiation Of Myelin-Producing OligodendrocytesDuring Developmental Myelination

Primary glial cell cultures were prepared from the dorsal telencephalonof newborn (P1) male mice as previously described in Feutz et al, J.Neurocytol, “Isolation and characterization of defective jimpyoligodendrocytes in culture”, 24:865-877 (2001). Briefly, meninges areremoved, and the dorsal telencephalons are microdissected andmechanically dissociated in DMEM supplemented with 10% calf serum,penicillin (50 U/ml), and streptomycin (50 μg/ml) (Thermo FisherScientific, France). The cell suspension is plated in a 24-well platescontaining 0.5 ml of cultured medium coated with 30 μg/ml poly-1-lysine(Sigma-Aldrich). Cultures containing astrocytes, oligodendrocytes (OLs)and microglia cells are then incubated in 5% CO2 and 95% air in ahumidified atmosphere (90%) at 37° C.

At 5 days in vitro (DIV), the culture medium of mixed primary glialcells was replaced by fresh medium supplemented with one of (i) the Smoagonist SAG (0.1 or 1 μM), (ii) SANT-1 (0.1 μM), (iii) testosterone (T,1 μM), (iv) SAG (0.1 or 1 μM) and testosterone (1 μM), (v) SANT-1 (0.1μM) and testosterone (1 μM), or (vi) the drug carriers as control. Thesupplemented culture medium was replaced every other day with a freshsolution. At 12 DIV, the cells were fixed for 20 minutes with 4%paraformaldehyde (PFA) in PBS, then permeabilized with 0.025% TritonX-100 for 10 minutes and blocked for 1 hour with Sea Block buffer(Thermo Fisher Scientific).

The cells were immunostained for BrdU and Olig2. For immunostaining,after overnight incubation with the primary antibodies at 4° C. followedby 3 washes in PBS, the cells were incubated with the appropriatesecondary antibodies for 2 hours before washing with PBS and theaddition of Fluoromount (Vector, clinisciences, France) as the mountingmedium. Images were acquired with immunofluorescence microscopy asdescribed above. (Data not shown). Quantification of Olig2+ BrdU+ wasevaluated by analyzing 3-4 independent cultures for each test condition,and these results are reported in FIGS. 2A-2B.

FIG. 2A shows the quantification of Olig2⁺ cells which have incorporatedthe proliferation marker BrdU 2 hours before the end of the culture, andindicates a synergic effect with SAG (0.1 μM) and testosterone (1 μM).FIG. 2A also show that in the absence of the active agents, Olig2⁺ BrdU⁺proliferating glial cells represent 2.0±0.4% of the total number ofOlig2⁺ cells, while the percentage of proliferating cells wassignificantly increased compared to control with SAG (1 μM) ortestosterone (1 μM), reaching 8.4±0.5 (p=6.93E-09) and 3.8±0.6% (p=0.04)of total Olig2 cells, respectively.

To analyse differentiation of myelin producing cells, the primary mixedglial cells were derived from Plp-EGP mice and cultured as describedabove. Detection of PLP+ cells co-expressing MBP was performed by usingprimary mixed glial cells derived from Plp-EGP mice and immunostainingfor Mbp. Immunofluorescence images were acquired as described above andthe number of PLP⁺ cells co-expressing MBP were evaluated. FIG. 2B showsthat SANT and testosterone administered together to primary mixed glialcells induce significantly higher levels of MBP expression than thatinduced by each active agent used alone (p=0.001). The immunostainingdata (not shown) reveal that testosterone (1 μM) and SANT (0.1 μM)highly increase the number of plp⁺GFP⁺ cells that co-express Mbp. TheSmo agonist SAG (0.1 μM or 1 μM) did not modify Mbp expression when itwas used alone. However, both concentrations of SAG induced a slight butsignificant decrease of testosterone-mediated OL differentiation.Remarkably, testosterone and SANT-mediated differentiating effectsappear to be additive when the drugs are used together. The inhibitionof Smo by SANT (0.1 μM) or testosterone (T, 1 μM) led to a 4-foldincrease in the percentage of PLP⁺ cells co-expressing MBP as comparedto the control condition (12.4±0.8 for SANT1, p=6.09E-07 and 11.7±1.5for testosterone, p=0.0005).

FIG. 2B further shows that when SANT and testosterone are used together,a 7-fold increase is observed in the percentage of mature OLs ascompared to the control (22.3±2.1 vs 3.0±0.9; p=2.22E-06). On the otherhand, the Smo agonist SAG (0.1 μM or 1 μM) did not modify Mbp expressionwhen it was used alone. However, both concentrations of SAG induced aslight but significant decrease of testosterone-mediated OLdifferentiation. Remarkably, testosterone and SANT-mediateddifferentiating effects appear to be additive when the drugs are usedtogether. While not being bound by theory, these results suggest thatthe hedgehog signaling blockade potentiates testosterone-inducedmaturation of oligodendrocyte progenitor cells (OPCs) into MBP+myelinating OLs.

To further investigate whether SANT+T promotes differentiation ofmyelin-producing oligodendrocytes during developmental myelination, P3male pups were subcutaneously treated with the SANT and/or testosteroneevery other day (n=3-5 animals per group) from the third to the tenthpostnatal day. SANT was used at a concentration of 20 μg/g pup weight,while testosterone was used at 20 μg for each administration. The drugswere injected subcutaneously every other day from postnatal day 3. AtP10, the pups were deeply anesthetized and perfused with PFA 4%. Thebrain was removed, post-fixed for 1 hour in PFA 4% and cryopreserved insucrose 30% before freezing and cryostat sectioning (14 μm).Subsequently, immunostaining for Olig2, Olig2/APC, Olig2/PDGFRα, andMBP/NF200 of the brain slices was performed at the level of thesubventricular zone and the adjacent corpus callosum (cc) of postnatalday 10 male pups treated with the Smo agonist SAG, the Smo antagonistSANT, and the steroid hormone testosterone (T). Images were acquiredwith immunofluorescence microscopy as described above (data not shown),and the different cell populations were quantified as shown in FIG.3A-3D.

FIG. 3D shows that combined treatment of testosterone and SANT-1promoted the production of myelin sheaths in male pups at P10. In theabsence of the active agents, the area occupied by MBP (the myelinatedarea) corresponded to 62.4±2.2% of the total area occupied by the axonsexpressing the neurofilament protein NF200. See FIG. 3D. In the presenceof SANT or testosterone, the percentage of myelinated area reached asignificantly higher value of 75.1±4.3 (SANT) (p=0.04) and 78.0±4.1% (T)(p=0.01). See FIG. 3D. When SANT and testosterone were concomitantlyinjected into the pups, the area occupied by the myelinated NF200⁺ MBP⁺axons reached 88.9±2.7%, a value significantly greater than obtainedwith SANT or testosterone alone (p=0.03 vs SANT, p=0.05 vstestosterone).

Thus, these data indicate that the Hedgehog and the androgen signalingpathways functionally interact during the neonatal wave ofoligodendrocyte precursor cell production, and during the subsequentdifferentiation of these cells into myelinating oligodendrocytes.

Overall, the results indicate that the combination SAG and testosteronepromotes proliferation of oligodendrocyte precursor cells but SAGinhibits testosterone-induced maturation of these cells intomyelin-producing oligodendrocytes.

Example 2 Smo Activation by SAG Promotes Production of New MatureOligodendrocytes and Precocious Increase of Pro-Regenerative Microgliain the LPC Model of Demyelination.

The effect of Smo activation by the Smo agonist SAG on demyelination wasinvestigated as follows. LPC-induced demyelination was performed aspreviously described in Ferent et al., J. Neuroscience 33:1759-1772(2013), in the absence or presence of SAG, in young male adult mice.Briefly, demyelinating lesions were induced unilaterally by stereotaxicinjections of 2 μl of a solution containing LPC 1% (Sigma-Aldrich) alongwith SAG (0.2 μM) or the corresponding vehicle, into the right corpuscallosum by using a Hamilton syringe specifically dedicated for neuralsurgery (NH BIO, France). The injection was performed at the followingcoordinates (to the bregma): anteroposterior (AP) +1 mm, lateral +1 mm,dorsoventral (DV) −2.2 mm. The brain was removed from deeplyanesthetized mice and transcardially perfused with 4% PFA. The tissuewas post-fixed for 4 hours in a fresh 4% PFA solution before beingcryopreserved in 30% sucrose, frozen in liquid nitrogen and cryostatsectioned (14 μm). 4-5 mice were used per time point for each treatmentcondition. Mice were sacrificed at 2 and 10 days post-lesion (dpl), andprepared for immunostaining of PDGFRα, Olig2/APC, Ki67/PDGFRα, GFAP, andIba1/Arg1. Images of immunostaining were acquired usingimmunofluorescence microscopy (data not shown), and the results werequantified by evaluating number of cells per surface unit for PDGFRa(FIG. 4A), Olig2/APC (FIG. 4B), Ki67/PDGFRa (FIG. 4C), GFAP (FIG. 4D),Iba1/Arg1 (FIG. 4E)

Remarkably, it was found that at 10 dpl SAG-treated animals displayed a2-fold increase in PDGFRα+ cells compared to the controls (121±11 vs65±5, p=0.008; FIG. 4A). Furthermore, the densities of Olig2+ APC+mature OLs were found to be significantly higher in the presence of SAGthan in the control condition (83+7 vs 59±1, p=0.027, FIG. 4B). Sincethe proportion of mature OLs appeared not significantly differentbetween the SAG-treated and control animals, it appears that SAG doesnot likely promote OPC differentiation.

At the earlier time point (2 dpl), new OPCs start to be recruited andhighly proliferate in the lesion while the tissue already displays ahigh inflammatory level. It was found that SAG treatment caused anincrease in the densities of proliferating Ki67+ PDGFRα+ OPCs comparedto the control condition (39±2 vs 28±2, p=0.007, FIG. 4C, left panel).Interestingly, the total number of PDGFRα+ OPCs remained unchangedindicating that SAG induced OPCs to enter the cell cycle. FIG. 4C, rightpanel.

To investigate a possible effect on the inflammatory cells, astrocytesand microglia were analyzed at the same time point. The area occupied byGFAP+ astrocytes tended to be decreased in the presence of SAG althoughnot in a significant manner. See FIG. 4D. Iba1+ microglia was found tobe unaffected by the presence of SAG as well. However, it was found thatthe density of the sub-population of microglia called “pro-regenerative”and characterized by the expression of Arginase-1 (Arg1) was enhanced by2-fold in the SAG-treated mice (103±7 vs 54±8, p=0.035, FIG. 4E, leftpanel). Furthermore, the proportion of Iba1 microglia co-expressing Arg1was increased two-fold by SAG treatment (58±5 vs 25±2, p=0.017; FIG. 4E,right panel). These results indicate that Hedgehog signaling activationby Smo agonists promotes the pro-regenerative potential of activatedmicroglia.

Since testosterone promotes astrocyte recruitment during remyelination,we investigated whether testosterone controls the phosphorylation ofsignaling transducer and activator of transcription 3(STAT3), which is amediator of signaling pathways important for myelin regeneration inastrocytes. The quantification of phosphorylated STAT3-expressingastrocytes was performed in brain slices derived from castrated malemice demyelinated by stereotaxic injection of lysolecithin after 7 daysof treatment with (1) intranasal administration of testosterone (T) at0.2 mg/day, (2) intranasal testosterone together with fadrozole (Fad)administered per oral route at 260 μg/kg, or (3) intranasal estradiol(E2) at 0.05 mg/day (n=4 per group). At 7 days post-lesion in theLPC-induced demyelination model, testosterone increased the number ofGFAP⁺ astrocytes expressing phosphorylated STAT3. The latter isimportant for both remyelination and astrocyte reactivity, which issimilar to activated microglia involved in local inflammation. Thiseffect appeared to involve testosterone aromatization to estradiolbecause the effect of testosterone could be inhibited by fadrozole, aninhibitor of the aromatase enzyme, as shown in FIG. 6. Moreover,estradiol also could increase the number of phosphorylatedSTAT3-expressing astrocytes to a similar extent as testosterone, asshown in FIG. 6.

Therefore, SAG and testosterone appeared to promote the pro-regenerativephenotypes of distinct inflammatory cells, microglia and astrocytes,respectively. Such complementary activities may likely be additive fordecreasing local inflammation.

Example 3 SAG+T Mitigates Experimental Autoimmune Encephalomyelitis.

Castrated male mice at age of 9-10 weeks were maintained for one weekfor acclimatization prior to experimental autoimmune encephalomyelitis(EAE) (n=10 animals per condition). The pathology was induced accordingto the instructions from the provider (Hooke Laboratories, MA, USA).Briefly, mice were immunized by subcutaneous injection of an emulsion ofMOG₃₅₋₅₅ peptide (myelin oligodendrocyte glycoprotein/MBP fragment35-55) in complete Freund's adjuvant (at two sites using 0.1 ml of thepre-conditioned mixture for each site) followed on the same day (Day 0)by a first intraperitoneal injection of pertussis toxin in PBS and by asecond one on Day 1 (250 ng/dose). The mice were scored blindly once aday starting at Day 7 post-immunization until Day 30 according to thefollowing scale: 0.0=no obvious changes in motor function; 0.5=tip oftail is limp; 1.0=limp tail; 1.5=limp tail and hind leg inhibition;2.0=limp tail and weakness of hind legs or signs of head tilting;2.5=limp tail and dragging of hind legs or signs of head tilting;3.0=limp tail and complete paralysis of hind legs or limb tail withparalysis of one front and one hind leg; 3.5=limp tail and completeparalysis of hind legs and animal unable to right itself when placed onits side; 4.0=limb tail, complete hind leg and partial front legparalysis with minimal moving and feeding; 4.5=complete hind and partialfront leg paralysis with no movement around the cage with the animalappearing no more alert; 5.0=extreme paralysis which requires euthanasiaof the animal.

The mice that developed EAE were randomly assigned into vehicle, SAG,testosterone or SAG+testosterone treatment groups in order to constitutegroups with similar time of EAE onset and similar onset scores (n=10animals per group). The active agents were administered at the onset ofclinical symptoms until Day 30 after immunization. Testosterone wasadministered via the intranasal route (0.2 mg/day in a volume of 2.5 μlin each nostril) in an oleogel composition as described above. SAG wasadministered orally by gavage (15 μg/g of mouse weight) every other day.

The animals were sacrificed by ketamine overdose. The spinalcord/vertebrae was removed and lumbar spinal cord/vertebrae samples weretreated according to the requirements of the various histologicalprocedures. For immunostaining, the spinal cord/vertebrae fragments werepost-fixed in PFA 4% for 24 hrs. The spinal cords were removed from thevertebral column, processed in an ethanol/xylene bath and embedded inparaffin blocks. 7-μm sections were then obtained using a microtome(Leica) and allowed to dry on glass slides overnight at 37° C. Forelectron microscopy, the spinal cord/vertebrae fragments were post-fixedin a mixture of PFA 2% and glutaraldehyde 2% for 5 days. The spinalcords were removed from the vertebral column, post-fixed incacodylate-buffered 1% osmium tetroxide for 1 hour at 4° C. and in 2%uranyl acetate for 1 hour at room temperature, and then dehydrated byserial dilutions of ethanol and embedded in epoxy resin. Ultrathinsections were contrasted with a saturated uranyl acetate solution.

FIG. 5A shows the improved clinical scores observed after treatment withboth SAG and testosterone, as compared to treatment with each activeagent separately. At days 18-19, animals in the three groups reached thescore 1.0 maintained until day 21 for each treatment. Between days 21and 30, testosterone-treated animals showed a relapse and the clinicalscore reached a value close to 2.0. In contrast, SAG-treated animalswere stabilized around the significantly lower score of 1.0. Remarkably,the drug combination fluctuated between the lowest clinical scores 0.5and 1.0, suggesting improved clinical scores by the combination of SAGand testosterone compared to using these drugs alone. See FIG. 5A.

To investigate the mechanisms involved in mitigating EAE, we examinedthe myelin levels and axon pathology at 30 days post EAE induction ineach condition. Electron microscopy images showed that the spinal cordsfrom the animals treated with SAG and testosterone alone or inassociation had much higher density of myelinated axons, and abnormalstructures were only occasionally observed. See FIG. 5B. Next, shows theg-ratio (axon diameter/total outer diameter of the myelinated fiber) ofat least 300 small calibre axons (≤2.5 m) per animal (n=3 for eachcondition) was determined and shown in FIG. 5C. In the spinal cordsderived from the control EAE animals, the g-ratio value (0.834±0.004)was significantly higher than the values determined for the animalstreated by testosterone (0.773±0.003, p<10⁻¹⁰), SAG (0.737±0.005,p<10⁻¹⁰) or the drug combination (0.743±0.005, p<10⁻¹⁰). Interestingly,FIG. 5C further shows that the administration of SAG alone or inassociation with testosterone led to a significantly lower g-ratio thantestosterone alone (p<10⁻⁵); whereas, the effect of SAG alone was notsignificantly different from the effect induced by the drug combination.Then, the numbers of abnormal structures (as described above) wereevaluated. See FIG. 5D. Higher percentages of abnormal axons weredetected in the spinal cord of the control EAE animals (36.4±4.0) thanin the testosterone (15.8±1.4, p=0.0004), SAG (14.0±1.7, p=0.0002) orSAG+testosterone (11.8±2.6, p=0.0001) treated mice.

In conclusion, SAG and testosterone administered separately orsubstantially simultaneously promote functional recovery associated withregeneration of myelin and neuroprotection.

Since the various effects observed for the combination treatment tendedto be higher than the effects of SAG alone, the consequences of theactive agents on the microglial cells was investigated. Spinal cordslices from EAE animals were immunostained with Iba1 and Arg1antibodies, which allowed the visualization of the whole activatedmicroglia and its pro-regenerative phenotype, respectively. Images wereacquired using immunofluorescence microscopy (data not shown), and theactivated microglia area was quantified and visualized as histograms inFIG. 5E-F. It was found that the control EAE mice had a high amount ofactivated Iba1⁺ microglia present in the spinal cord, but these cellswere not polarized towards their pro-regenerative phenotype. Neithertestosterone nor SAG was found to significantly modify the total densityof activated microglia in the spinal cord as shown in FIG. 5E. However,SAG (10.9±1.7, p=0.03) but not testosterone (6.8±1.4) promoted microgliapolarization towards the Arg1+ anti-inflammatory and pro-regenerativephenotype as compared to control (4.8±0.5) as shown in FIG. 5F.Unexpectedly, activated microglia collapsed as a whole compared to thecontrol when the active agents were used simultaneously (1.8±0.2,p=0.03, FIG. 5F), indicating that the combination appears to solve apathological microglia activation.

In order to assess the effects of testosterone and SAG alone or incombination in astrocytes, spinal cord from EAE animals were alsoimmunostained with GFAP antibody. Quantifications performed inimmunofluorescent microscopy images indicated that testosterone and SAGtend to increase or decrease GFAP-positive area, respectively. TheGFAP-positive area in EAE animals treated with testosterone, SAG, or thecombination of testosterone and SAG was not significantly different fromthe GFPA-positive area in the control group. However, testosterone(38.8±1.8) induced a significantly higher GFAP staining than SAG(25.1±2.2, p=0.001). The co-administration of testosterone and SAG ledto a GFAP-positive area comparable to the GFAP-positive area in thecontrol group as shown in FIG. 5G, suggesting that testosterone and SAGmay regulate different subsets of GFAP-positive astrocytes potentiallyinvolved in the beneficial effects of the combination therapy.

Since lymphocyte recruitment into the brain across vascular endothelialcells of the blood brain barrier represents an important event in thepathogenesis of the EAE model and multiple sclerosis itself, the effectsof testosterone and SAG used alone or in combination on the permeabilityof the blood brain barrier were assessed. To assess the permeability ofthe blood brain barrier, an antibody directed to the tight junctionprotein Claudin 5 was used, since the proteins of this family confer toendothelial cells an ability to strictly regulate the passage of solubleand cellular elements between the blood and the central nervous system.Quantifications performed in immunofluorescent microscopy imagesindicated that testosterone (2,53±0.09, p<0.0001), SAG (2.35±0.27,p<0.0001) and the combination therapy (2.03±0.15, p<0.0003) induced ahighly significant increase in the expression of Claudin 5 compared tothe control condition (0.65±0.11) with no significant difference betweenthe treatments, as shown in FIG. 5H. Thus, testosterone and SAG usedalone or in combination appear to exhibit a beneficial activity towardsrestoring the efficiency of the blood brain barrier.

Overall, the results indicate that combination treatment with an Smoagonist and a steroid hormone has synergistic therapeutic effects in oneof the most relevant models for MS.

1-16. (canceled)
 17. A method of reducing inflammation in the brain in asubject in need thereof, comprising administering to the subject aSmoothened (Smo) agonist.
 18. The method according to claim 17, whereinthe Smo agonist is3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide.19. The method according to claim 17, wherein the method furthercomprises administering to the subject testosterone.
 20. The methodaccording to claim 19, wherein the Smo agonist and testosterone areadministered in separate compositions, substantially simultaneously orsequentially.
 21. The method according to claim 19, wherein the Smoagonist and testosterone are administered in the same composition. 22.The method according to claim 17, wherein the Smo agonist isadministered intranasally in an intranasal pharmaceutical compositionthat further comprises (a) at least one lipophilic or partly lipophiliccarrier present in an amount of from about 60% to about 98% by weight ofthe formulation; (b) at least one compound having surface tensiondecreasing activity present in an amount of from about 1% to about 20%by weight of the formulation; and (c) at least one viscosity regulatingagent present in an amount of from about 0.5% to about 10% by weight ofthe formulation.
 23. The method according to claim 22, wherein theintranasal pharmaceutical composition further comprises testosterone.24. The method according to claim 17, wherein the Smo agonist isadministered intranasally in an intranasal pharmaceutical compositioncomprising a porous excipient, wherein the Smo agonist is loaded ontosurfaces of the porous excipient located inside pores of the porousexcipient.
 25. The method according to claim 19, wherein both the Smoagonist and testosterone are administered intranasally in an intranasalpharmaceutical composition comprising a porous excipient, wherein theSmo agonist and testosterone are loaded onto surfaces of the porousexcipient located inside pores of the porous excipient.
 26. The methodaccording to claim 17, wherein the subject is a human, a non-humanprimate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or arat.
 27. The method according to claim 17, wherein the subject suffersfrom multiple sclerosis, amyotrophic lateral sclerosis, or Alzheimer'sdisease.
 28. The method according to claim 17, wherein administering theSmo agonist promotes microglia polarization towards an Arg1+anti-inflammatory phenotype.
 29. The method according to claim 19,wherein administering both the Smo agonist and testosterone reducesmicroglia activation.